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- PDB-5nv3: Structure of Rubisco from Rhodobacter sphaeroides in complex with CABP -
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Open data
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Basic information
Entry | Database: PDB / ID: 5nv3 | ||||||
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Title | Structure of Rubisco from Rhodobacter sphaeroides in complex with CABP | ||||||
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Function / homology | ![]() ![]() ![]() ![]() Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ![]() ![]() ![]() | ||||||
![]() | Bracher, A. / Milicic, G. / Ciniawsky, S. / Wendler, P. / Hayer-Hartl, M. / Hartl, F.U. | ||||||
![]() | ![]() Title: Mechanism of Enzyme Repair by the AAA Chaperone Rubisco Activase. Authors: Javaid Y Bhat / Goran Miličić / Gabriel Thieulin-Pardo / Andreas Bracher / Andrew Maxwell / Susanne Ciniawsky / Oliver Mueller-Cajar / John R Engen / F Ulrich Hartl / Petra Wendler / Manajit Hayer-Hartl / ![]() ![]() Abstract: How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) ...How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair. | ||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 906.6 KB | Display | ![]() |
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PDB format | ![]() | 775.1 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 3699MC ![]() 3700C ![]() 3701C ![]() 3702C M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 51768.852 Da / Num. of mol.: 8 / Fragment: RbcL Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() References: UniProt: P27997, ![]() #2: Protein | Mass: 15183.234 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() References: UniProt: P27998, ![]() #3: Sugar | ChemComp-CAP / #4: Chemical | ChemComp-MG / |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: Rubisco![]() | ||||||||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() ![]() | ||||||||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() Details: RcaCC hexamers (20 micromolar monomer) were mixed with E.C.M-CABP octamers (10 micromolar monomer) in a reaction containing 20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM ATP and 1mM ...Details: RcaCC hexamers (20 micromolar monomer) were mixed with E.C.M-CABP octamers (10 micromolar monomer) in a reaction containing 20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM ATP and 1mM RuBP, for 1 min at 25oC prior to addition of 0.125 % of glutaraldehyde (GA). After 10 min the reaction was quenched by addition of 0.1M Tris HCl pH 8 followed by gel filtration on a Superdex 200 PC 3.2/30 column (GE Healthcare).The fractions were eluted in buffer A and analyzed on a 6 % native gel. Fraction 13 containing HMW complexes with the least amount of free Rubisco were chosen for cryo-EM. The crosslinked E.C.M.-CABP-RcaCC complexes were diluted to 0.0030-0.0035 g ml-1 in 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM ATP, 1 mM ATP-gammaS and 1 mM RuBP | ||||||||||||||||||||||||||||||
Vitrification![]() | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: Cs corrected Krios 1 at NeCEN (June 2016) |
Electron gun | Electron source![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() |
Image recording | Average exposure time: 1.25 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) |
Image scans | Movie frames/image: 22 |
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Processing
EM software |
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CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Symmetry | Point symmetry![]() ![]() | ||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 333122 / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: RECIPROCAL / Target criteria: Maximum likelihood |